POS0334 LYSYL OXIDASES (LOX(L)) PROMOTE PATHOLOGIC CHONDROCYTE CALCIFICATION
Authors: I. Bernabei, M. Romani, E. Faure, J. Wegrzyn, T. Hügle, N. Busso, S. Nasi
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Pathologic calcification (PC) is the deposition of calcium-containing crystals in soft tissues. PC of cartilage is a hallmark of osteoarthritis (OA), a degenerative joint disease characterized by cartilage degradation. The interplay between calcification- and extracellular matrix (ECM) formation, maturation and degradation in cartilage is not fully understood. <h3>Objectives</h3> We aim to reveal the role of lysyl oxidases (LOX(L)), a family of extracellular enzymes that catalyze collagen crosslinking, in OA cartilage calcification. <h3>Methods</h3> We induced <i>in vitro</i> chondrocytes calcification with calcifying medium (CM = BGJb +10% FBS+ 20mM β-glycerophosphate + 50μg/ml ascorbic acid) or control (Nt = DMEM high glucose + 10% FBS), and treated or not with 500μM β-aminoproprionitrile (BAPN). Gene expression was analyzed by qRT-PCR, Il-6 in cell supernantants by ELISA, mitochondrial and cytoplasmic reactive oxygen species (ROS) by use of the kits MitoSOX and DHE (Life Technologies). We induced i<i>n vivo</i> murine knee calcifications using the menisectomy (MNX) mode, where medial meniscus is removed in the right knee, while the left knee is sham operated. During two months, mice were injected with PBS or BAPN 50mg/Kg twice per week, and given 1mg/ml BAPN in drinking water. New calcific formation in knees were analyzed by microCT. Bioinformatics analysis was performed with an online tool at www.systems-genetics.org/mmad, using the Modules by Module section in GeneBridge. Manhattan plots were generated by correlating collagen fibril organization module (GO:0030199, representing LOX/L family of enzymes, and calcification-, chondrocyte hypertrophy- and catabolic enzymes-associated modules. <h3>Results</h3> Our findings revealed that CM and calcium-containing crystals induced lox(l) activity in murine chondrocytes <i>in vitro</i>. Conversely, treatment with the lox(l) pan-inhibitor BAPN, decreased crystal production in vitro, while decreasing calcification gene expression of <i>Anx5</i>, <i>PC1</i>, <i>Pit-1/2</i> and Alp activity. Intriguingly, we identified that the pro-calcifying effects of LOX/L were mediated via their classical cross-linking activity, as diminished cross-links in pre-formed extracellular matrix reduced in parallel chondrocyte calcification, with specific suppression of <i>Anx5</i> and <i>Pit-1/2</i> genes. Additionally, BAPN reverted the hypertrophic phenotype of calcifying chondrocytes, reducing expression of <i>Runx2</i> and <i>Col10</i> and increasing that of early differentiation <i>Sox9</i> and <i>Col2</i>. In the same conditions, LOX/L inhibition downregulated <i>Col1</i> and <i>Col3</i>, whose uncontrolled production can lead to ECM fibrosis and calcification. BAPN also inhibited the expression and release of pro-calcifying cytokine Il-6, while also reducing mitochondrial and cytoplasmic ROS, known to be associated with PC. Additionally, we revealed in calcifying chondrocytes that BAPN inhibition decreased cartilage catabolic enzymes genes (<i>Mmp3</i>, <i>Mmp13</i> and <i>Adamts5</i>). In line, treatment with BAPN in an <i>in vivo</i> murine OA model based on meniscectomy, led to decreased knee joints calcifications, while not affecting the bone mineral density of the knee. We also confirmed the role of lox/l in human OA cartilage calcification. Indeed. LOX(L) inhibition by BAPN led to reduced crystal production in primary human OA chondrocytes. Finally, a bioinformatics transcript analysis of available human and murine databases obtained from tissues potentially undergoing PC, further confirmed a positive association between LOX(L)/collagen fibril organization module and calcification-, chondrocyte hypertrophy- and catabolic enzymes-associated modules. <h3>Conclusion</h3> Collectively, our results revealed for the first time a pro-calcifying effect of LOX(L) in chondrocytes, through classical (cross-linking of extracellular matrix) and non-classical pathways (i.e. chondrocyte hypertrophy, increased matrix synthesis and degradation, and exacerbated IL-6 and ROS production). In conclusion, LOX(L) provide a novel target for future therapies aimed at treating pathologic cartilage calcification. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.